ATPbased Assay

1
ATP-based Assay

• ATP is
• used as energy source in all living forms
• a molecule linked to evidence of life
• fairly stable
• ATP-bioluminescence assay
• ATP transfers its energy to the molecule
  luciferin. The reaction is catalyzed by the
  presence of the enzyme luciferase and the
  magnesium ion. The luciferin emits light. The
  intensity of the bioluminescence generated is
  directly proportional to the amount of ATP in the
  sample being assayed.
• Rapid, quantitative, and commercially available
• Kikkoman Corp. kit differentiates free
  extra-cellular (dead cells, etc.) from
  intra-cellular (viable microbes) ATP
• When supplemented with conventional bacterial
  assays, able to distinguish the presence of
  non-cultivable microorganisms from cultivable
  microorganisms
• Currently used in the food industry

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2
Extracellular vs Viable cells
Microorganisms
Removal of extracellular ATP
ATP
ATP
Extracellular ATP
ATP
ATP
ATP
ATP
ATP
ATP
ATP
ATP
ATP
ATP
ATP
ATP releasing reagent
Bioluminescence reagent
Overestimation
ATP from viable cells
ATP from both viable and dead cells
False result
High precision
Low sensitivity
9
3
ATP content in various microbes
Relative luminescene unit
One Relative Luminescence Unit (RLU) 1 cell
11
4
Relationship between Colony Counts and
Intracellular ATP
ATP standard
10-7 10-8 10-9 10-10 10-11 10-12
10-13
10-7
10-9
ATP concentration (M)
10-11
Aureobasidium pullulans
10-13
101
103
105
RLU
Bacillus sp.
Intracellular ATP (M)
Staphylococcus epidermidis
Pseudomonas stutzeri
Bacillus pumilus spores
102 103 104
105 106
107 108 109
12
Colony counts (CFU/mL)